2/2/2012. What I will talk about. Second generation sequencing using. SNP genotyping and sequencing service. Uppsala University

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1 Second generation sequencing using HiSeq2000 and enome Sequencer FLX Ulrika Liljedahl Manager sequencing applications SNP&SEQ technology platform, Dept of Medical Sciences, Uppsala University What I will talk about he SNP&SEQ technology platform Introduction to sequencing using the HiSeq2000 and enome Sequencer FLX+ technologies pplications of second generation sequencing, projects at the SNP&SEQ technology platform SNP&SEQ technology platform, Uppsala University Founded 2001, sequencing since beginning of 2008 Located at kademiska sjukhuset, enter for linical i l and Medical lresearch, entrance 70 Make modern genotyping and sequencing technology available to researchers SNP genotyping and sequencing service ccredited according to ISO/IE17025 Illumina SPro for genotyping and sequencing In house developed LIMS to keep track of samples, plates, genotypes, flowcells etc eniospro (ecan) SNPstream BeadExpress (Beckmanoulter) BeadStation iscan K 5M No of SNPs HiSeq2000 enome Sequencer FLX+ (Roche/454) 1

2 SNP genotyping on all scales Staff as of January 2012 Instrumentation single SNPs to 5 million per sample enome wide arrays: Illumina 700K, OmniExpressExome,1M, 2.5M and 5M Focused content human: ardiometabo200k, Immuno200K, Exomehip Focused content: Bovine700K, anine170k, Ovine70K, Porcine50K, MouseLinkage ustom arrays up to 200,000 SNPs for any organism pplications: ssociation studies, opy Number Variation, Methylation ti profiling, Evolutionary and population genetics omas xelsson PhD, amilla Enström orbjörn Öst Ingvar horsteinsson nn-hristin Wiman Dana Magnúsdóttir Kjell Stålberg Ulrika Liljegren roup leader SNP Res. engineer Res. engineer Res. engineer Res. engineer Res. engineer Res. engineer Res. engineer nn-hristine Syvänen Ulrika Liljedahl, PhD, Kristina Larsson Marie Lindersson Pia Lindström Johanna Lagensjö Karin Sollander Helena Fällmar manda Raine Professor roup leader SEQSenior res. engineer Senior res. engineer Res. engineer Res. engineer Res. engineer Res. engineer Res. engineer Olof Karlberg, PhD Lillebil ndersson roup leader Bioinfo dmin. assistant eniospro (ecan) SNPstream (Beckmanoulter) BeadExpress BeadStation iscan ommy Hellberg I syst admin Henrik Ljungberg I syst & data security Per Lundmark Bioinfomatician Jonas arlsson Edvard Englund, PhD Bioinfomatician Database developer K 5M No of SNPs Lars Bäckström roup leader Isyst Second generation sequencing HiSeq2000 enome Sequencer FLX+ (Roche/454) MiSeq (Illumina, available mid March 2012) HiSeq2000, Illumina Read length HiSeq2000 Run time days Output Upgrade HiSeq , 100 or 150bp Single read or paired end 1.4 billion reads 270 bases sequence data per F with PE bases in 27h or standard run HiSeq2000 enome Sequencer FLX+ (Roche/454) 2

Library preparation Ligation of adapter sequences

Starting material: DN RN, cdn Enriched regions of

library from 1µg DN 24 barcodes ruseq 48 barcodes

Exon enrichment ruseq or SureSelect S t(gilent) RN

barcodes ScriptSeq (Epientre) Matepair long insert

3 Library preparation Ligation of adapter sequences to the fragments. Barcoding possible. Starting material: DN RN, cdn Enriched regions of interest dapter 0 ypes of sequencing libraries DN library from 1µg DN 24 barcodes ruseq 48 barcodes NextFlex (Bioo Scientific) Morein custom panels Exon enrichment ruseq or SureSelect S t(gilent) RN library from 1µg totalrn 24 barcodes ruseq 12 barcodes ScriptSeq (Epientre) Matepair long insert 2 5kb, not a good protocol from Illumina smallrn 48 barcodes ruseq 3

4 luster generation Flowcell with 8 lanes (channels) dapter ligated fragments hybridised to flowcell channels Isothermal lbid bridge amplification to generate clusters Performed in cbot instrument During imaging, each lane divided into 3 swaths and each swath divided into 8 sections (tiles). Lane Swath ile 4

5 5

6 luster generation leaving a cluster with forward strands only. 6

he HiSeq2000 instrument ouch screen Syringe pumps Sequencing By Synthesis (SBS) 3 5 ycle 1: dd sequencing reagents First base incorporated Optics: Remove unincorporated bases 532nm laser + 660nm

7 he HiSeq2000 instrument ouch screen Syringe pumps Sequencing By Synthesis (SBS) 3 5 ycle 1: dd sequencing reagents First base incorporated Optics: Remove unincorporated bases 532nm laser + 660nm laser + 785nm laser focus Waste bottle 4 Ds, one per base Flow cell Detect signal ycle 2-n: dd sequencing reagents and repeat ll four labelled nucleotides in one reaction Single read vs paired-end Runtime 2-11 days Keyboard and mouse tray Reagents compartment 5 Results in up to 30bases per lane if PE100 High accuracy Base-by-base sequencing No problems with homopolymer repeats Sequence reads HiSeq 2000 Scanner Single read 5 3 Line scanner Read all four colors simultaneously sweeps per surface (2048 pixles/sweep) Paired-end Read1 50 or 100bp Index read 6bp Read2 Both upper and lower surface imaged 8 tiles per sweep 48 tiles per lane Every tile processed separately Read1 Index read 5 Upper surface (op) Lower surface (Bot) lusters 7

8 Software 100 MIRONS 1 BILLION LUSERS PER FLOW ELL 20 MIRONS HiSeq ontrol Software HS Fluidics and scanning Identifies clusters luster Intensity File (IF) Real ime nalysis R Identifies bases (Basecalling) alculate base quality Error rate spike in control sample Move data to server Base all File (bcl) Statistics nalysis workflow Summary HiSeq bases from one flowcell Relatively short reads bp heap per base Barcodes enable sample multiplexing oo much data for some applications 8

9 MiSeq Flowcell with one lane Runtime 27h 2x250bp read length (coming spring2012) 7bases per run (coming spring2012) On instrument analysis enome Sequencer FLX+ Roche 454 sequencing 9

Summary S FLX+ Fast run time 23h 500 700bp long reads (75 150bp

using SOLiD or HiSeq) Junior system available 35Mb data, 400bp

Sequencing technology and applications HiSeq 2000 Illumina enome

entire genomes arget regions RN-sequencing (transcriptome

10 Summary S FLX+ Fast run time 23h bp long reads (75 150bp for SOLiD and HiSeq) 700Mbases from one run ( b per flowcell using SOLiD or HiSeq) Junior system available 35Mb data, 400bp read length High run costs empr quite messy to work with Sequencing technology and applications HiSeq 2000 Illumina enome Sequencer FLX+ Roche/454 Re-sequencing or de-novo sequencing of entire genomes arget regions RN-sequencing (transcriptome profiling, expression, smallrn etc) hip-seq to study DN-protein interactions Methylation 10

11 Organisms and research areas over the years, genotyping and sequencing lzheimer disease stma QL mapping Bipolar disease Expression Linkage map ancer QL mapping Linkage map Inbreeding analysis VD depression Speciation QL mapping Re-sequencing Linkage map Osteoporosis Sequencing QL mapping Speciation Sjögrens syndrome Sequencing SLE Stroke Suicidiology ranscriptome enome 2D sequencing Sequencing Sequencing, cancer enome and genetics transcriptome sequencing Mutation argeted gene screening sequencing Domestication Disease genetics enetic Lymph node Sequencing autoimmune Sequencing archaeology expression pathogenic disorders Expression argeted gene analysis funghi analysis sequencing Speciation enetic archaeology Breeding evolution hank you for your attention! ny questions? 11

Overview of Next Generation Sequencing technologies. Céline Keime

Overview of Next Generation Sequencing technologies Céline Keime keime@igbmc.fr Next Generation Sequencing < Second generation sequencing < General principle < Sequencing by synthesis - Illumina < Sequencing